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. 2023 Feb 20;5:1130736. doi: 10.3389/fgeed.2023.1130736

FIGURE 4.

FIGURE 4

T-CAST analysis for TRAC-targeting TALENs (A) Genotyping. Results of T7E1 assay of WT-TALEN and OH-TALEN (KKR-ELD and KVR-EAD configuration) are shown. 32°C indicates that T cells were subjected to transient cold-shock after electroporation, while control cells were constantly cultured at 37°C. * specifies p-value<0.05 (Student’s t-test, n = 3–6). (B) TCR expression. Displayed is the fraction of TCRα/β-negative T cells upon transfer of TALEN-encoding mRNA as determined by flow cytometry. Where indicated (32°C) T cells were subjected to a transient cold-shock. *** specifies p-value<0.001 (Student’s t-test; n = 3–7). (C–E) Structural variations. Circos plots illustrate T-CAST results with enlargement of the chromosome 14 region encompassing TRAC. Red lines represent chromosomal rearrangements (OMTs with >20 hits) with the TRAC target site. (F) Genotyping. NGS at denoted sites was performed on untreated (UT) T cells, and T cells edited with WT-TALENs or OH-TALENs as indicated. The group in which the samples were originally identified is indicated on the bottom. *** specifies p-value<0.001 (Fisher’s exact test, Bonferroni corrected p-values).